# 1104: DETHIOBACTER MEDIUM


## Main sol. 1104

6	g	NaCl	
0.5	g	K2HPO4	
8	g	NaHCO3	
22	g	Na2CO3	
1.5	g	Sulfur (powdered)	
0.2	g	NH4Cl	
0.2	g	MgCl2 x 6 H2O	
1	ml	**Trace elements solution (Pfennig & Lippert,1966)**	
1	ml	**Selenite-tungstate solution**	
0.05	g	Yeast extract	
0.6	ml	Ethanol absolute	
0.4	g	Na-acetate	
2.5	g	Na2S2O3 x 5 H2O	
1	ml	**Wolin's vitamin solution (10x)**	
0.24	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve sodium chloride and hydrogenphosphate, then sparge solution with 100% N2 gas for 30 -45 min to make it anoxic. Add and dissolve carbonates, then dispense under 100% N2 gas atmosphere into anoxic Hungate-type tubes or serum vials containing already the appropriate amount of sulfur, only to 30% of their volume to allow for a large headspace. Sterilize medium by heating cultivation vessels in a water bath to 90 - 100°C for 1- 2 hours on each of 3 successive days. After sterilization add ammonium chloride, magnesium chloride, trace elements, yeast extract, ethanol, acetate, thiosulfate, vitamins and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas. The stock solutions of thiosulfate and vitamins should be sterilized by filtration. Adjust pH of the complete medium to 9.5.
2. After inoculation add sterile 100% H2 gas to 0.5 bar overpressure.


## Trace elements solution (Pfennig & Lippert,1966)

5	g	EDTA	
2.2	g	FeSO4 x 7 H2O	
0.1	g	ZnSO4 x 7 H2O	
0.03	g	MnCl2 x 4 H2O	
0.03	g	H3BO3	
0.2	g	CoCl2 x 6 H2O	
0.03	g	CuCl2 x 2 H2O	
0.03	g	NiCl2 x 6 H2O	
0.03	g	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. pH 3.0-4.0


## Selenite-tungstate solution

0.5	g	NaOH	
3	mg	Na2SeO3 x 5 H2O	
4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	


## Wolin's vitamin solution (10x)

20	mg	Biotin	
20	mg	Folic acid	
100	mg	Pyridoxine hydrochloride	
50	mg	Thiamine HCl	
50	mg	Riboflavin	
50	mg	Nicotinic acid	
50	mg	Calcium D-(+)-pantothenate	
1	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
50	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water